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. 2023 Mar 29;18(3):e0283749. doi: 10.1371/journal.pone.0283749

Axl contributes to efficient migration and invasion of melanoma cells

Hanshuang Shao 1,2, Diana Teramae 1,2, Alan Wells 1,2,*
Editor: Suzie Chen3
PMCID: PMC10057740  PMID: 36989239

Abstract

Axl, a member of the TAM receptor family has been broadly suggested to play a key role in tumor metastasis. However, the function of Axl in the invasion and metastasis of melanoma, the most lethal skin cancer, remains largely unknown. In the present study, we found that melanoma cell lines present variable protein levels of Axl and Tyro3; interestingly, MerTK is not noted at detectable levels in any of tested MGP (metastatic growth phase) cell lines. Treatment with recombinant human Gas6 significantly activates Akt in the Axl-expressing WM852 and IgR3 lines but just slightly in WM1158. IgR3, WM852 and WM1158 demonstrate different autocrine signaling. Knockdown of Axl by siRNA or the treatment with Axl-specific inhibitor R428 dramatically inhibits the migration and invasion of both IgR3 and WM852 in vitro. These findings suggest that Axl enhances the invasion of melanoma cells.

Introduction

Melanoma originating from the pigmented melanocytes in the basal layer of the epidermis is the most lethal skin cancer although it accounts for only 1% of all patients with skin malignancies [1, 2]. Primary melanoma can be successfully surgically excised if the tumor cells grow radially. However, the 5-year relative survival rates of patients with melanoma dramatically decrease once the disseminated tumor cells invade into and especially through the dermis. Thus, vertical invasiveness is a harbinger of poor outcomes for these patients.

The overall prognosis of patients has dramatically improved in the past decades due to the availability of early screens, advanced surgeries, targeted therapy, and immune checkpoint inhibitors [3, 4]. For example, specific inhibition of BRAF leads to a strong response and survival benefit in most cases. However, only a small fraction of patients with advanced melanoma can achieve long-term survival [5, 6]. The major reason is likely due to the acquisition of drug resistance and in the survival signaling from the metastatic tumor microenvironment. For example, the activation status of PTEN controls the AXL/AKT axis mediated-resistance to BRAF inhibitor (BRAFi) [7]. Therefore, determining the functions of the key proteins which lead to the initial invasion, of melanoma is still urgent and necessary.

TAM receptor family of tyrosine kinases consists of Axl, Tyro3 and MerTK. Among three members, Axl is the most studied and has been shown to be elevated in many cancers including melanoma, breast, lung, ovary, pancreas, and prostate [8–12]. Axl can be activated when its ligand, growth arrest-specific protein (Gas6), binds on resulting in a cascade of downstream signaling pathways [13]. Gas6 consists of three major domains [14]. Gas6/Axl mediates migration and invasion in many cancer types such as breast cancer, prostate cancer, and osteosarcoma [15–17]. We previously revealed that Tyro3 is important in melanoma migration via its phosphorylation and role in the regulation of ACTN4 [18]. In this study, we queried whether Axl could contribute to melanoma invasiveness.

Materials and methods

Reagents and cell culture

Validated Axl (Cat #: 4390824) and scramble siRNA (Cat #: 4390843), Transfection reagents Lipofectamine RAN/MAX 2000 (Cat #: 13778–150) and Lipofectamine 2000 (Cat #: 11668–019) were purchased from Life Technologies (Grand Island, NY). Axl (Cat #: TF320269 set A-D) and scramble (Cat #: TR30007) shRNAs were purchased from Origene (Rockville MD). Recombinant hGas6 (Cat #: 885-GSB) was purchased from R & D system (Minneapolis MN). Recombinant human epidermal growth factor (EGF) (Cat #: E9644-.2MG) was purchased from Sigma (St. Louis, MO). Axl inhibitor R428 (Cat #: A8329) was purchased from ApexBio (Houston TX). G418 (Cat #: G5005), was purchased from Teknova (Hollister CA). Antibodies including EGFR (Cat #: 4267S), pEGFR(Y1173) (Cat #: 4407S), pAkt(S473) (Cat #: 3787S), pERK (Cat #: 4370S), pP38 (Cat #:4511S), GAPDH (Cat #: 5174S), pAxl (Y702) (Cat #: 5724S) were purchased from Cell Signaling Technology. Monoclonal antibodies against Axl (Cat #:sc-166269), Tyro3 (Cat #: sc-166359) and ACTN4 (Cat #: sc393695) were purchased from Santa Cruz Biotechnology (Dallas, TA). MatriGel 24w transwells (Cat #: 354480) were purchased from Corning (Corning NY). Melanoma cell line IgR3, WM852, WM1158, WM983A, WM983B, FEMX and A375 were cultured in a medium containing 3 parts of DMEM (1gL-1 glucose) (Cat #: 10-014-CV) and 1 part of Leibovitz’s 15 (L15) (Cat #: 11415–064, Gibco, Billings, MT) with 10% fetal bovine serum (Cat #: 100–016, GeminiBio, West Sacramento, CA) and 1x pen/strep antibiotics (Cat #: 15140–122, Gibco). Melanoma cell line 1025Lu were maintained in RPMI 1640 (Cat #: 61870–036, Gibco) medium with 10% fetal bovine serum and 1x pen/strep antibiotics. Melanocytes were grown in DermaLife M media (Cat #: LM-0004) in the presence of growth factors and chemical components (Cat #: LS-1030, Lifeline Cell Technology, Frederick, MD).

Immunoblotting

Cells grown in 6-well tissue culture plates were transfected with siRNA or treated with Gas6 or EGF prior to lysing in RIPA buffer containing 1x protease inhibitors cocktails set V. The lysate was incubated on ice for 5 min prior to sonicating briefly. Samples were collected to microcentrifugation tubes and then centrifugated at 13,000g at 4°C for 30 min. After transferring the supernatant to new tubes, the concentration of total soluble proteins of each sample was determined using BCA™ Protein Assay (Cat #: 23227, Thermo Scientific™ Pierce™, Rockland, IL). To run a SDS gel, ten micrograms of total proteins from each sample was mixed with one fifth volumes of 5x SDS sample buffer with β-mercaptoethanol to get a running sample containing 1x SDS sample buffer. Protein samples were denatured by boiling for 3 min and then applied to SDS gel with different concentrations of acrylamide based on the molecular size of interest protein. Then, separative proteins in SDS gel were transferred to polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with 5% fat free milk at room temperature for half followed by incubation with indicated primary antibodies in cold room overnight. Next day, the membrane was washed 3 times with 1x TBS buffer containing 0.1% Tween 20 for 5 min each. Then the membrane was incubated with secondary antibody at room temperature for 45 min followed with throughout washing. Finally, interest proteins on PVDF membrane were developed using enhanced ECL reagents.

Transfection and selection of stable cell lines

In a six-well plate, 4x105 cells were seeded in each well and cultured in complete growth medium overnight to get about 60% cell density at transfection. Next day, the medium was replaced with fresh Opti-medium with low serum followed by an addition of a complex of Lipofectamine RNA/MAX and siRNA for 4h incubation. Then, cells were incubated at 37°C in a humidified incubator with 5% CO2 for 48h prior to further. For selecting stable expressing cells, transfected cells were further cultured in complete growth medium containing 1400 μg/ml G418 until monoclonal colonies were big enough to be picked up.

In vitro scratch wound assay

Confluent cells grown in six-well plate were slowly scratched with a rubber scraper to create a wound area at about 3 mm in width. After completely washing away the floating scratched cells, two lines vertically with scratched edge at the bottom of each well were marked. Then, images involving each mark line were taken as time 0h. After 24h incubation, images at same positions were retaken. For the treatments of Gas6 and EGF, cells were starved with quiescence media containing 0.5% dialyzed fetal bovine serum overnight prior to scratching. Finally, the width of each wound along the mark lines area was measured using Photoshop software. The relative migratory rates were the wide difference between time 0h and 24h.

Transwell invasion assay

Cells were detached from culture plate with trypsin treatment and collected into a 15 ml Falcon tube. After centrifugating at 250g for 5 min, cells were resuspended in growth medium at a density of 50,000 cells/ml. Then, half a milliliter of cells was applied to a well equilibrated MatriGel transwell for an additional 48h incubation. For Gas6 treatment, cells were starved in quiescence medium containing 0.5% dialyzed FBS overnight and appropriate amount of Gas6 was added to the quiescence medium in the bottom chamber of transwell. After aspirating the medium in transwell chamber, all cells retaining on the top surface of MatriGel were removed using a loosen cotton swab. Then, cells invaded through the MatriGel and membrane and attached on the other side of membrane were fixed followed by staining with 0.5% crystal violet or permeabilized using Triton X-100 and staining with DAPI. Finally, cells were imaged and counted at different area under microscope.

Results

TAM and EGFR family receptor profiles of melanoma cell lines

We, and others have previously reported that melanoma cell migration is promoted by signals through receptors belonging to the EGFR/HER and TAM families [18–20]; These receptors have been implicated in metastatic progression across many different tumor types by driving tumor cell motility that enables invasion and dissemination. Vertical migration through the dermal layer is the critical step in developing metastatic melanoma.

To understand the signals that impel primary melanoma tumors to become metastatic, we determined the expression of TAM family members (Axl, Tyro3 and MerTK) in melanoma cell lines including vertical growth phase (VGP) and metastatic growth phase (MGP) using immunoblotting. Compared to melanocytes, all eight melanoma cell lines present increased expression of Tyro3 (Fig 1). Interestingly, the Axl level in four out of five MGP cell lines (except for WM983B) is significantly elevated but only one cell line IgR3 from VGP shows more Axl expression. MerTK, a lesser studied TAM member, is not readily detectable in melanoma cell lines except in two VGP cell line 983A and FEMX. As there has been reported associations between Axl and EGFR family members and this has been proposed as a means to overcome resistance to anti-EGFR therapy [20–24], we queried the protein expression level of EGFR/HER family. As shown in Fig 1, EGFR is dramatically upregulated in four MGP cell lines (except for WM983B) and one VGP cell line IgR3 which are all Axl positive. ErbB2 is slightly increased in all tumor cell lines except for a significant increase in WM852 and undetectable in 1205Lu, similar to negligible if any levels in melanocytes. Surprisingly, all three VGP cell lines present a significant increase in ErbB3 expression while two MGP cell line WM1158 and 1205Lu do not express any ErbB3. The expression of ErbB4 is detectable in all eight cell lines but at low levels requiring extensive exposure times. Interestingly, while the expression pattern appears to be cell line specific, the more invasive and metastatic lines present upregulation of Axl and EGFR (except for WM983B), which appears absent in the VGP lines, with the exception of IgR3.

Fig 1. TAM and EGFR family receptor profiles of melanoma cell lines.

Fig 1

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VGP and MGP melanoma cells were lysed in RIPA buffer and soluble proteins were separated by SDS-PAGE for immunoblotting against indicated antibodies. Shown are representative blots of three experiments.

As we have reported that melanoma invasion involves the cytoskeleton to membrane linker protein alpha-actinin-4 [19] and that this is a downstream target of both receptor families [18, 25, 26], we catalogued its level. We found that this target is elevated in the eight cancer cell lines queried. The levels in the MGP lines were somewhat higher than in the VGP lines with the exception of IgR3 again expressing levels similar to MGP lines.

Axl-dependent activation of intermediary signaling pathways by Gas6

Axl has been shown to play a key role in the acquisition of therapeutic drug resistance via activation of intermediary survival signals including the PI3kinase-Akt pathway [22]. These same pathways are involved in progression related cell behaviors including cell locomotion [27] and cell phenotype differentiation [28]. We queried whether the TAM-binding growth-arrest specific protein (Gas6) triggers differential activation of intermediary signaling pathways in different cell lines. We selected IgR3 and WM852 from VGP and MGP groups, respectively, as these lines express more Axl, Tyro3 and all four HER family members. We treated both IgR3 and WM852 cells, both starved in quiescence media containing 0.1% dialyzed FBS overnight, with recombinant Gas6 at a concentration of 200 ng/ml for 10 min. As shown in Fig 2A, we detected significant phosphorylation of Axl at Y702 in both IgR3 and WM852 which resulted in downstream activation of Akt. This is consistent with data found by [16, 17]. p38 is just slightly activated in WM852 and not at all in IgR3 when Gas6 was applied. Surprisingly, we did not observe an increase in pERK in either IgR3 or WM852 in response to Gas6. Of note, there may be some autocrine signaling as IgR3 presents higher basal level of pAkt than WM852 (NT lane). On the other hand, WM852 shows high basal p38.

Fig 2. Gas6-mediated activation of Axl in melanoma cells.

Fig 2

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(A) Indicated melanoma cell lines were transiently transfected with control or Axl siRNA for 2 days prior to starving in quiescence media for 16h. Quiescent cells were then stimulated with indicated factors at the concentrations of 10 nM (EGF) and 200 ng/ml (Gas6) for 10 min followed by lysis in RIPA buffer for immunoblotting using the indicated antibodies. (B) Immunoblotting of indicated proteins extracted from quiescent melanoma cell lines treated with or without 200 ng/ml Gas6 for 10 min. NT: non treatment. Shown are representative blots of three experiments.

As Axl has been shown to interact with EGFR in breast cancer cells and glioblastoma multiformes [23, 29], we were interested in whether Gas6 can activate EGFR in both IgR3 and WM852 cells in which both Axl and EGFR are highly upregulated. We did not observe phosphorylation of EGFR at Y1173 in WM852 cells and negligibly at best in IgR3 cells; the EGFR was functioning as shown by increased phosphorylation when exposed to 10 nM EGF for 10 min. Combination of Gas6 and EGF did not enhance pEGFR in WM852 and but did so significantly in IgR3.

To determine whether Gas6 activates Axl specifically and not other TAM family receptors, we downregulated Axl expression using siRNA (Fig 2A). Knockdown of Axl eliminated Gas6-mediated activation of Akt in both cell lines. To further confirm Gas6 specifically activates Axl, we applied Gas6 on another melanoma cell line WM983B in which Axl is undetectable. As shown in Fig 2B, pAkt levels are not affected by application of Gas6 to WM983B compared to IgR3 and WM852. This result also suggests Gas6 activation in IgR3 and WM852 is not through Tyro3 as WM983B expresses more Tyro3 than IgR3 and WM852.

Gas6 enhances the migration and invasion of melanoma cells

The above data show that Gas6 leads to activation of intermediary signal pathways that contribute to cell migration. Next, we were interested in whether Gas6 promotes the migration of melanoma cells. As growth media contains stimulatory growth factors, we optimized the amount of dialyzed FBS and Gas6 that could demonstrate a contribution of signaling through Axl. As shown in Fig 3A, we observed a significant activation of Akt by Gas6 in all treatments. Although the lower concentration of FBS (0.1%) presented trace of pAkt in the absence Gas6, the contents of both pAxl and pAkt in the presence of Gas6 were also lower than those in other treatments. High concentration of FBS such as 2% led to the highest activation of Axl and Akt but the untreated (NT) cells showed higher basal activation of Axl and Akt. Therefore, we chose 0.5% FBS for optimizing the concentration of Gas6. We found that the phosphorylation levels of both Axl and Akt were gradually enhanced when increasing the concentration of Gas6 (Fig 3B).

Fig 3. Gas6 promotes migration and invasion of melanoma cells in vitro.

Fig 3

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(A) Immunoblotting of indicated proteins extracted from WM852 cells quiesced in quiescent media containing indicated percentage of dialyzed FBS and stimulated with 300 ng/ml Gas6 for 15min. (B) WM852 cells were quiesced in 0.5% FBS quiescent media for 16h and then stimulated with indicated concentration of Gas-6 for 15min. Immunoblottings were performed using indicated antibodies. Shown are representative of three experiments. (C) Quantitative results of relative migratory speed of indicated melanoma cell lines stimulated with indicated factors for 24h. (C) Quantitative results of invaded IgR3 and WM852 with or without an attraction of 500 ng/ml Gas6 for 48h. Data are mean of ± SD of three independent experiments. Statistical analysis was performed using Student’s t-test. * P < 0.05, NS: not significant.

We incubated melanoma cells in quiescence media containing 0.5% dialyzed FBS with 500 ng/ml of Gas6 for 24h to determine the effect of Gas6 on the migration of melanoma cells. As shown in Fig 3C, Gas6 dramatically promoted the migration of WM852, while moderately lifting the migration of IgR3 though IgR3 cells present higher level of migration in the absence of added Gas6. Migration induced by EGF was a positive control and showed that Gas6 was as active as EGF in driving motility. Interestingly, Gas6 and EGF were not additive. As a negative control, 983B cells showed no increase in motility by either Gas6 or EGF, as these cells do not express appreciable levels of either receptor Axl or EGFR (Fig 1). These findings suggest that Axl plays an important role in Gas6-mediated enhanced migration of IgR3 and WM852 (Fig 2A).

To further determine if Gas6 promotes invasion of IgR3 and WM852, we placed IgR3 or WM852 cells starved with 1.0% FBS quiescence media on the top of MatriGel and allowed the cells invade for 48h. We increased the concentration of dialyzed FBS in this assay due to extended time exposure and to optimize cell attachment and invasion through the MatriGel (S1 Fig). As we expected, 500 ng/ml Gas6 plus 1% FBS dramatically promoted the invasions of both IgR3 and WM852 (Fig 3). These data provide a role for Gas6 in promoting melanoma invasion through a matrix barrier consistent with prior findings suggesting such a role [16, 17, 30].

Axl contributes to intrinsic migration and invasion

As Axl is abundant in IgR3 and WM852 cells and high density of receptors with intrinsic tyrosine kinase activity can lead to ligand-independent activation [31–34], we queried whether Axl contributes to the migration and invasion of these two cell lines even in the absence of Gas6. We transiently transfected these two cell lines with validated siRNA prior to performing monolayer wound scratch and MatriGel transwell invasion assays. The Axl siRNA dramatically downregulated the expression of Axl in both IgR3 and WM852 cells while the protein level of Tyro3 was not affected by Axl siRNA providing for the specificity of the siRNA (Fig 4A).

Fig 4. Transient silencing of Axl using siRNA reduces migration and invasion of melanoma cells.

Fig 4

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IgR3 and WM852 cells were transiently transfected with Axl siRNA for 48h. (A) Immunoblottings were performed using indicated antibodies to show selective downregulation of Axl. (B) Quantitative results of relative migratory speed of transfected cells with or without 1 μM of R428 treatment. (C) Representative images of DAPI-stained cells invaded through MatriGel and retained on the bottom of transwell membrane. (D) Quantitative results of invaded cells. Shown are representative blots and pictographs of three experiments. Data are mean of ± SD of three independent experiments. Statistical analysis was performed using Student’s t-test. * P < 0.05, NS: not significant.

Axl siRNA significantly limited the migration of IgR3 and WM852 cells (Fig 4B, NT, black columns). To attribute this effect to Axl signaling, we observed that R428, an Axl-selective inhibitor at a concentration of 1 μM significantly reduced the migration IgR3 and WM852 cells transfected with control siRNA, but only slightly if at all cells in which the siRNA downregulated Axl. The reduced cell motility of both IgR3 and WM852 cells was not due to the possibility of R428 reducing cellular viability (S2 Fig). WM852 were almost completely killed in the presence of 10 μM of R428 for 24hr while IgR3 presented partial survival (S3A Fig). This difference in tolerating high concentration of R428 was probably due to the different level of activated Akt (pAkt at S473) even in the absence of added stimuli (Fig 2A and 2B, “NT” & “-”lanes). The basal level of pAkt in IgR3 was significantly higher than that in WM852. Axl siRNA did not affect the basal level of pAkt in IgR3 which explains why Axl siRNA did not increase the sensitivity of IgR3 cells to R428 (S3B Fig). In contrast, Axl siRNA made WM852 more sensitive to low concentration of R428 such as 2.5 μM and 5 μM (S3B Fig).

Next, we performed a MatriGel transwell invasion assay to determine whether Axl siRNA and R428 affect the invasion of IgR3 and WM852 cells. As shown in Fig 4C and 4D, both Axl siRNA and R428 dramatically reduced the number of invaded cells. The combination of Axl siRNA and R428 was not additive suggesting that R428 functioned specifically via Axl receptor. Taken together with prior reports on Axl functioning [35, 36], Axl is required for migration and invasion of melanoma IgR3 and WM852 cells in vitro.

To further confirm the role of Axl in migration and invasion in melanoma cells, we constructed a set of Axl shRNA vectors to generate stable cell lines of IgR3. After selection in puromycin, we obtained several stable mono-clones in which Axl was dramatically downregulated, but Tyro3 expression was not affected (Fig 5A). Monolayer scratch assays showed that all Axl stable clones migrated significantly slower compared to clones made with a scramble shRNA (Fig 5B). The invasion of all stable clones was dramatically less than cells transfected with the scramble shRNA clone (Fig 5C and 5D). These data further confirm that Axl is a key component for enhanced migration and invasion of melanoma cell line IgR3.

Fig 5. Stable knockdown of Axl limits migration and invasion of IgR3 cells.

Fig 5

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(A) Immunoblottings of stable anti-Axl shRNA expressing IgR3 monoclonal clones using indicated antibodies show selective downregulation of Axl. (B) Quantitative results of relative migratory speed of scramble and Axl shRNA clones. (C)Representative images of DAPI-stained cells invaded through MatriGel and retained on the bottom of transwell membrane. (D) Quantitative results of invaded cells. Shown are representative blots and pictographs of three experiments. Data are mean of ± SD of three independent experiments. Statistical analysis was performed using Student’s t-test. * P < 0.05, ** P< 0.01, NS: not significant.

Discussion

The lethal event in melanoma progression is invasion through the dermis, allowing for dissemination of the tumor cells to distant organs. The drivers of such would thus be targets to prevent the further dissemination of secondary melanomas after initial diagnoses. As we have shown that tyrosyl-directed phosphorylation of ACTN4 regulates amoeboid invasion of melanoma cells [19], we have sought the upstream regulators of this signaling pathway. As TAM family members lead to phosphorylation of ACTN4 [18], we queried whether the member most commonly associated with tumors [12], Axl, could drive melanoma progression.

In a survey of melanoma-derived cell lines we found that Tyro3 was ubiquitously present, but high levels of Axl were selective for metastatic growth phase (MGP) lines, with the exception of IgR3 (Fig 1). Thus, we investigated the role of Axl in these cells. IgR3 cells were originally isolated from a patient carrying a vertical growth phase melanoma tumor [37]. We found that stimulation of Axl with ligand could drive motility and transmigration of a barrier matrix. Finally, knockdown of Axl in IgR3 cells dramatically limited its migration and invasion in vitro. Unfortunately, we failed to get stable Axl shRNA WM852 cell lines when we selected it using puromycin. We noted that WM852 cells transfected with Axl shRNA grew very slowly and gradually died during selection implying that Axl is required for the viability of WM852 cells. This also suggests that Axl functions variably on the viability in each melanoma cell line.

Given that Axl can trigger the activation of Akt, ERK and p38, we tested in this study if silencing Axl by siRNA limits the basal activation of these signaling molecules. Axl siRNA did not abolish the basal activation of Akt, ERK and p38 in the absence of Gas6 stimulation but eliminated ligand-activated activation of Akt (Fig 2A). Axl has been suggested to be involved in the interaction of melanoma cells and tumor microenvironment during metastasis [12]. Thus, silencing Axl could affect these interactions resulting in an impaired invasion and metastasis. The microphthalmia transcription factor (MITF) plays a vital role in melanoma cells [5]. Previous studies revealed that most of melanoma cell lines present MITFlow/Axlhigh [5, 38]. Knockdown of Axl might affect the expression of MITF-dependent key proteins in IgR3 cells.

Our data are consistent with those of others that show that knockdown of Axl reduced the invasion of additional melanoma lines [4, 39, 40], lending further support to the contention that Axl can be target to control melanoma progression. This is in addition to the upstream targets that others have identified such as YAP [4]. In aggregate the data shown herein, demonstrating a role for Axl in promoting migration and invasion, argue for motility signaling as being key to metastatic dissemination. Thus, targeting Axl, in addition to other growth factor signaling pathways (including Braf and EGFR) [5, 41] could improve therapy aimed at preventing melanomas from metastasizing and generating lethal tumors.

Supporting information

S1 Fig. FBS enhances the invasion of IgR3 cells.

Quiesced IgR3 cells were invaded in quiescence media containing indicated concentration of FBS for 24h. Representative images are nuclei of invaded cells stained with DAPI.

(DOCX)

Click here for additional data file. (138.6KB, docx)
S2 Fig. Low concentration (1uM) of R428 has no effect on the viability of melanoma cells.

Representative images of IgR3 and WM852 cells transfected with either control or Axl siRNA and treated with or without 1 μM of R428 for 24h. Shown are random images from each well.

(DOCX)

Click here for additional data file. (491.4KB, docx)
S3 Fig. IgR3 and WM852 demonstrate variable tolerance to R428.

(A) Representative images of IgR3 and WM852 cells treated with indicated concentration of R428 for 24h. (B) Representative images of IgR3 and WM852 cells transfected with either control or Axl siRNA and treated with indicated concentration of R428 for 24h. Images were randomly taken from each well.

(DOCX)

Click here for additional data file. (803.3KB, docx)
S1 File

(PDF)

Click here for additional data file. (625.2KB, pdf)

Acknowledgments

We thank the members of the Wells lab for suggestions.

Data Availability

All relevant data are within the paper and the original blots are supplied in the Supporting information file as per PLoS ONE guidance.

Funding Statement

This work was supported by a Merit Award from the Veterans Administration (BX003368 to AW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Suzie Chen

1 Feb 2023

PONE-D-22-30461Axl contributes to efficient migration and invasion of melanoma cellsPLOS ONE

Dear Dr. Wells,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript by Shao and colleagues describes investigation into the effect of AXL stimulation and level on the migration and invasion of melanoma cells. The authors demonstrate clearly that stimulation of Axl signalling with Gas6 increases melanoma migration and invasion in lines with Axl expression. Further, knockdown of Axl expression either using transient or stable means reduces melanoma migration and invasion, as does treatment with a specific inhibitor. While the data arising from the stable knockdown is novel, much of the other data presented in the manuscript is confirmatory.

The manuscript overall would benefit from careful revision and editing. The experiments appear to have been conducted carefully with sufficient replication evident from the description. However, the authors should acknowledge the previous work of others in the manuscript on the topic at hand:

Sensi M, et al. (2011). "Human cutaneous melanomas lacking MITF and melanocyte differentiation antigens express a functional Axl receptor kinase." J Invest Dermatol 131:2448–57.

Use of shAXL to reduce invasion in matrigel/boyden chamber

Use of R428 to reduce migration and invasion

Tworkoski, K., et al. (2011). "Phosphoproteomic screen identifies potential therapeutic

targets in melanoma." Mol Cancer Res 9(6): 801-812.

Use of shAXL to reduce migration

Pietrobono S, Anichini G, Sala C, Manetti F, Almada LL, Pepe S, Carr RM, Paradise BD, Sarkaria JN, Davila JI, Tofani L, Battisti I, Arrigoni G, Ying L, Zhang C, Li H, Meves A, Fernandez-Zapico ME, Stecca B. ST3GAL1 is a target of the SOX2-GLI1 transcriptional complex and promotes melanoma metastasis through AXL. Nat Commun. 2020 Nov 17;11(1):5865. doi: 10.1038/s41467-020-19575-2.

Use of shAXL to reduce invasion in matrigel/boyden chamber

Zhang X, Yang L, Szeto P, Abali GK, Zhang Y, Kulkarni A, Amarasinghe K, Li J, Vergara IA, Molania R, Papenfuss AT, McLean C, Shackleton M, Harvey KF. The Hippo pathway oncoprotein YAP promotes melanoma cell invasion and spontaneous metastasis. Oncogene. 2020 Jul;39(30):5267-5281. doi: 10.1038/s41388-020-1362-9. Epub 2020 Jun 19.

PMID: 32561850

Use of shAXL to reduce invasion in matrigel/boyden chamber

In summary, the data presented in Figure 2A and B, Figure 3A-D and Figure 4A-D has been previously published by others.

Figure 2A is currently very confusing. It would be best to group the western blot images by cell line rather than antibody. Further, the figure legend states 10 nM EGF was used whereas the results text states 2 nM. Which is correct?

There is currently no text in the results section that addresses the data in Figure 5.

The reference to figures throughout the manuscript needs revision. Further, the labelling of Supplementary Figure 2 A, B - A, B, C and D is confusing and should be revised.

Reviewer #2: Eight melanoma lines were evaluated for expression of EGFR and AXL family members. IgR3 and WM852 were then stimulated with Gas6 and EGF and evaluated for pAKT, pAXL, p38, pEGFR, and pERK. Knockdown of AXL blocked Gas6 induced pAKT in both lines.as well as a line lacking AXL (WM983b) although TYRO3 was present, indicating that AXL is a mediating of Gas6 induced Akt phosphorylation. WM852 showed stronger relative migration speeds induced by Gas6 or EGF compared to IgR3. With respect to invasion, IgR3 showed higher basal migration than WM852 but there was a similar absolute increase induced by Gas6. Basal migration and invasion of the two lines was reduced either by an AXL inhibitor or siRNA.

The main claims of the paper are that AXL can mediate motility and invasion of melanoma cell lines. This has not been demonstrated previously for melanoma and thus adds to the body of knowledge for this disease.

The authors should discuss the current knowledge regarding AXL mediating Gas6 induced migration/invasion in other tumor types. There are some concerns regarding selected claims noted below. Detailed protocols are not required but more details regarding specific reagents and the initial width in the scratch assay should be indicated. The manuscript is written clearly and well organized with no dual use concerns.

Other comments:

On page 5, there is reference to “all 4 MGP” and “all 4 VGP”. Figure 1 however does not clearly indicate 4 VGP lines – please correct the inconsistency. Is WM983B a VGP, MGP or neither?

In referring to Figure 2 on page 5, it is stated” Surprisingly, we did not observe an increase in pERK in either IgR3 or WM852.” However, the blot seems to clearly show increased pERK for IgR3 in response to EGF and possibly also for GM852. Similarly, though weaker, there is EGFR phosphorylation in response to EGF although it is claimed that there is not.

The statement on page 6 in the middle : “These findings indicate that Axl signaling is cell-line specific” is confusing – one possibly interpretation is there are lines that express axl but do not respond to Gas6. Please clarify or remove.

Catalog numbers for siRNAs, antibodies, transwells, growth media, growth factors (Gas6, EGF) and all other biological reagents should be provided.

For Figure 3, please indicate the absolute basal migration speeds for all the lines. Does the smaller relative increase for IgR3 simply reflect a higher basal migration speed?

The statement at the top of page 7 “…suggest that pAkt plays important role in Gas6-mediated enhanced migration of IgR3 and WM852” does not seem justified since no inhibitor of Akt phosphorylation was tested.

Figure S2 legend refers only to panels A and B while the figure is labeled A- D.

In the discussion it is stated that ASL siRNA eliminated Gas6 induced p38 phosphorylation – that does not seem supported by Figure 2 since there is little gas6 induced p38 phosphorylation, especially in IgR3. Quantitative analysis and statistics should be used to show this point.

**********

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Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2023 Mar 29;18(3):e0283749. doi: 10.1371/journal.pone.0283749.r002

Author response to Decision Letter 0

2 Mar 2023

Journal Requirements:

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Response: It is now removed from the manuscript. Please amend to: "This work was supported by a Merit Award from the Veterans Administration (BX003368 to AW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

4. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. "A preliminary version of this manuscript has been uploaded to bioRxiv at https://www.biorxiv.org/content/10.1101/2022.05.02.490307v1" Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published.

Response: The posted manuscript was neither peer-reviewed nor formally published.

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Response: Please state that all data are presented in the manuscript and the Supporting Information files.

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Response: Both cropped and the cognate uncropped blots are now provided in the Supporting Information files.

7. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Response: We now provide these data in the revised manuscript to show that the invasion of IgR3 in 1% FBS medium is significantly higher than that in 0.1% FBS medium.

8. Please ensure that you refer to Figure 5 in your text as, if accepted, production will need this reference to link the reader to the figure.

Response: We now address the data in Figure 5 in the revised text.

9. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Response: We have updated the references as suggested by the reviewers.

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript by Shao and colleagues describes investigation into the effect of AXL stimulation and level on the migration and invasion of melanoma cells. The authors demonstrate clearly that stimulation of Axl signalling with Gas6 increases melanoma migration and invasion in lines with Axl expression. Further, knockdown of Axl expression either using transient or stable means reduces melanoma migration and invasion, as does treatment with a specific inhibitor. While the data arising from the stable knockdown is novel, much of the other data presented in the manuscript is confirmatory.

Comment: The manuscript overall would benefit from careful revision and editing. The experiments appear to have been conducted carefully with sufficient replication evident from the description. However, the authors should acknowledge the previous work of others in the manuscript on the topic at hand:

Sensi M, et al. (2011). "Human cutaneous melanomas lacking MITF and melanocyte differentiation antigens express a functional Axl receptor kinase." J Invest Dermatol 131:2448–57.

Use of shAXL to reduce invasion in matrigel/boyden chamber

Use of R428 to reduce migration and invasion

Isolated melanoma cell lines

Tworkoski, K., et al. (2011). "Phosphoproteomic screen identifies potential therapeutic

targets in melanoma." Mol Cancer Res 9(6): 801-812.

Use of shAXL to reduce migration

Yale isolated melanoma cell lines

Pietrobono S, Anichini G, Sala C, Manetti F, Almada LL, Pepe S, Carr RM, Paradise BD, Sarkaria JN, Davila JI, Tofani L, Battisti I, Arrigoni G, Ying L, Zhang C, Li H, Meves A, Fernandez-Zapico ME, Stecca B. ST3GAL1 is a target of the SOX2-GLI1 transcriptional complex and promotes melanoma metastasis through AXL. Nat Commun. 2020 Nov 17;11(1):5865. doi: 10.1038/s41467-020-19575-2.

Use of shAXL to reduce invasion in matrigel/boyden chamber

A375 cell line and patient-derived melanoma cells. We actually cited this paper in ref #4

Zhang X, Yang L, Szeto P, Abali GK, Zhang Y, Kulkarni A, Amarasinghe K, Li J, Vergara IA, Molania R, Papenfuss AT, McLean C, Shackleton M, Harvey KF. The Hippo pathway oncoprotein YAP promotes melanoma cell invasion and spontaneous metastasis. Oncogene. 2020 Jul;39(30):5267-5281. doi: 10.1038/s41388-020-1362-9. Epub 2020 Jun 19.

PMID: 32561850

Use of shAXL to reduce invasion in matrigel/boyden chamber

Response: We apologize for these oversights. We now added these data and references with statement in the discussion.

Comment: In summary, the data presented in Figure 2A and B, Figure 3A-D and Figure 4A-D has been previously published by others.

Response: We include these data as part of the complete story as these are new data sets but refer to earlier works in the text. We used Gas6 to check if the interaction between Axl and EGFR is involved in the activation of Gas6-depependent of Axl in available melanoma cell lines. These data also confirmed that Gas6 functions in Axl positive melanoma cells.

Comment: Figure 2A is currently very confusing. It would be best to group the western blot images by cell line rather than antibody. Further, the figure legend states 10 nM EGF was used whereas the results text states 2 nM. Which is correct?

Response: We now reorganized Fig. 2A by cell line according to reviewer’s suggestion. For short stimulation of cells with EGF, we usually use a concentration of 10 nM. We mistyped the concentration of EGF in text but that is now corrected.

Comment: There is currently no text in the results section that addresses the data in Figure 5.

Response: We apologize for this oversight. We now address the data in Figure 5.

Comment: The reference to figures throughout the manuscript needs revision. Further, the labelling of Supplementary Figure 2 A, B - A, B, C and D is confusing and should be revised.

Response: Figure S2 now is reorganized, relabeled and renumbered as Figure S3 in the revised manuscript.

Reviewer #2: Eight melanoma lines were evaluated for expression of EGFR and AXL family members. IgR3 and WM852 were then stimulated with Gas6 and EGF and evaluated for pAKT, pAXL, p38, pEGFR, and pERK. Knockdown of AXL blocked Gas6 induced pAKT in both lines.as well as a line lacking AXL (WM983b) although TYRO3 was present, indicating that AXL is a mediating of Gas6 induced Akt phosphorylation. WM852 showed stronger relative migration speeds induced by Gas6 or EGF compared to IgR3. With respect to invasion, IgR3 showed higher basal migration than WM852 but there was a similar absolute increase induced by Gas6. Basal migration and invasion of the two lines was reduced either by an AXL inhibitor or siRNA.

The main claims of the paper are that AXL can mediate motility and invasion of melanoma cell lines. This has not been demonstrated previously for melanoma and thus adds to the body of knowledge for this disease.

Comment: The authors should discuss the current knowledge regarding AXL mediating Gas6 induced migration/invasion in other tumor types. There are some concerns regarding selected claims noted below. Detailed protocols are not required but more details regarding specific reagents and the initial width in the scratch assay should be indicated. The manuscript is written clearly and well organized with no dual use concerns.

Response: We thank the reviewer for the suggestion. We now discussed this in the part of discussion of our revised manuscript. The width of the wound scratch area is about 3 mm. It is now stated in the revised manuscript.

Other comments:

Comment: On page 5, there is reference to “all 4 MGP” and “all 4 VGP”. Figure 1 however does not clearly indicate 4 VGP lines – please correct the inconsistency. Is WM983B a VGP, MGP or neither?

In referring to Figure 2 on page 5, it is stated” Surprisingly, we did not observe an increase in pERK in either IgR3 or WM852.” However, the blot seems to clearly show increased pERK for IgR3 in response to EGF and possibly also for GM852. Similarly, though weaker, there is EGFR phosphorylation in response to EGF although it is claimed that there is not.

Response: We apologize for our imprecise statements. As the labeling in Figure 1, there are three VGP cell lines and five MGP cell lines; we now corrected this in the revised manuscript. For the reference to Figure 2 on page 5, we stated that “Surprisingly, we did not observe an increase in pERK in either IgR3 or WM852” referred to no significant pERK increase when cells are treated only by Gas6. The reviewer is correct in that EGF treatment significantly increased the pERK which has been mentioned on page 6. We also stated that “the EGFR was functioning as shown by phosphorylation when exposed to 10 nM EGF for 10 min” on page 6. This meant that the EGF treatment triggers the phosphorylation of EGFR in both IgR3 and WM852 cells. It is stronger in WM852 and weaker in IgR3. These statements are now clarified.

Comment: The statement on page 6 in the middle: “These findings indicate that Axl signaling is cell-line specific” is confusing – one possibly interpretation is there are lines that express axl but do not respond to Gas6. Please clarify or remove.

Response: We thank the review for pointing out this issue. We now deleted the statement in the revised manuscript.

Comment: Catalog numbers for siRNAs, antibodies, transwells, growth media, growth factors (Gas6, EGF) and all other biological reagents should be provided.

Response: We now provide catalog numbers for all reagents.

Comment: For Figure 3, please indicate the absolute basal migration speeds for all the lines. Does the smaller relative increase for IgR3 simply reflect a higher basal migration speed?

Response: The reviewer is correct in that IgR3 presents much high basal migration. We now updated Figure 3C with relative migratory units instead of normalized data to more clearly show this.

Comment: The statement at the top of page 7 “…suggest that pAkt plays important role in Gas6-mediated enhanced migration of IgR3 and WM852” does not seem justified since no inhibitor of Akt phosphorylation was tested.

Response: We thank the reviewer for pointing out this misstatement. We now corrected it to “suggest that Axl plays an important role in Gas6-mediated enhanced migration of IgR3 and WM852.

Comment: Figure S2 legend refers only to panels A and B while the figure is labeled A- D.

Response: We thank the reviewer for noting this omission. We now reorganized this figure and corrected the figure legend as shown in figure S3 in the revised manuscript.

Comment: In the discussion it is stated that ASL siRNA eliminated Gas6 induced p38 phosphorylation – that does not seem supported by Figure 2 since there is little gas6 induced p38 phosphorylation, especially in IgR3. Quantitative analysis and statistics should be used to show this point.

Response: We thank the reviewer for noting the lack of significance of this and have corrected this in the revised manuscript.

Attachment

Submitted filename: Response Letter R1.pdf

Click here for additional data file. (93KB, pdf)

Decision Letter 1

Suzie Chen

15 Mar 2023

Axl contributes to efficient migration and invasion of melanoma cells

PONE-D-22-30461R1

Dear Dr. Wells,

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Acceptance letter

Suzie Chen

20 Mar 2023

PONE-D-22-30461R1

Axl contributes to efficient migration and invasion of melanoma cells​

Dear Dr. Wells:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. FBS enhances the invasion of IgR3 cells.

    Quiesced IgR3 cells were invaded in quiescence media containing indicated concentration of FBS for 24h. Representative images are nuclei of invaded cells stained with DAPI.

    (DOCX)

    Click here for additional data file. (138.6KB, docx)
    S2 Fig. Low concentration (1uM) of R428 has no effect on the viability of melanoma cells.

    Representative images of IgR3 and WM852 cells transfected with either control or Axl siRNA and treated with or without 1 μM of R428 for 24h. Shown are random images from each well.

    (DOCX)

    Click here for additional data file. (491.4KB, docx)
    S3 Fig. IgR3 and WM852 demonstrate variable tolerance to R428.

    (A) Representative images of IgR3 and WM852 cells treated with indicated concentration of R428 for 24h. (B) Representative images of IgR3 and WM852 cells transfected with either control or Axl siRNA and treated with indicated concentration of R428 for 24h. Images were randomly taken from each well.

    (DOCX)

    Click here for additional data file. (803.3KB, docx)
    S1 File

    (PDF)

    Click here for additional data file. (625.2KB, pdf)
    Attachment

    Submitted filename: Response Letter R1.pdf

    Click here for additional data file. (93KB, pdf)

    Data Availability Statement

    All relevant data are within the paper and the original blots are supplied in the Supporting information file as per PLoS ONE guidance.

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